The effect of pH on the oxidation of bovine serum albumin by hypervalent myoglobin species

Bovine serum albumin (BSA) was used as a probe for the oxidation of proteins by hypervalent myoglobin species in solutions with pH from 5.3 to 7.7. The reaction between perferrylmyoglobin, *MbFe(IV)=O, and BSA was studied by activating metmyoglobin with equimolar amounts of hydrogen peroxide in the presence of BSA. A minor pH dependence was observed as judged from the formation of BSA-centered radicals, which were monitored at room temperature by electron spin resonance spectroscopy, and the formation of dityrosine. The reaction between ferrylmyoglobin, MbFe(IV)=O, and BSA was pH-dependent. BSA-centered radicals and dityrosine were formed in low levels at neutral pH and increased at low pH to the same levels as observed in the reaction of *MbFe(IV)=O with BSA. The present results demonstrate that protein-centered radicals can be formed from the non-radical MbFe(IV)=O under mildly acidic conditions, and this should be taken into account when considering oxidation in cellular compartments of low pH and in meat-related products.     

Binding of diclofenac sodium with bovine serum albumin at different temperatures, pH and ionic strengths

The study was designed to examine the binding of diclofenac sodium with bovine serum albumin (BSA) at different temperatures (20 degrees, 30 degrees and 40 degrees C), pH (6.4, 7.4 and 8.4) and ionic strengths (micro = 0.1, 0.2 and 0.3) by means of equilibrium dialysis method. The concentration of diclofenac sodium was maintained at wider range from 15 to 900 micromole/l and BSA concentration was maintained at 61.5 micromole/l. The data obtained were interpreted by nonlinear regression method using Graphpad prism software. The analysis showed that the interaction between diclofenac sodium with BSA results in two-site saturable binding. A decrease in association constant was observed with increasing temperature. The average standard free energy change (deltaGdegrees) value was -7.07 (site I) and -4.2 (site II) Kcal/mol. The standard enthalpy change (deltaHdegrees) and the standard entropy change (deltaSdegrees) were -7.8 Kcal/mole, -2.35 cal/mole (site I) and -7.4 Kcal/mole, -10.5 cal/mole (site II), respectively. The negative enthalpy change suggested the binding between diclofenac sodium and the binding sites of BSA were spontaneous and exothermic. The negative value of deltaHdegrees and deltaSdegrees indicated hydrogen bonding and van der Waal's force was the major mechanism for diclofenac sodium and BSA interaction. Increase in pH and ionic strength also caused decrease in association constant of diclofenac sodium and BSA binding.     

The role of pH and its control on effective conjugation of bovine hemoglobin and human serum albumin

Human serum albumin (HSA) and bovine hemoglobin (Hb) conjugate is a promising candidate as a blood substitute. However, preparation of the conjugate is problematic because both proteins tend to conjugate between themselves rather than crosslink each other. In this work, a facile process for conjugation of Hb and HSA was developed through control strategy of the reaction. The reaction was carried out in a buffer containing borax-borate and mannite. The borax-borate was used for pH buffering while mannite was used as a pH switch and a reaction promoter. As a result, self-conjugation of Hb and self-conjugation of HSA were minimized. After the one-step conjugation reaction in aqueous solution, followed by the one-step purification by ion-exchange chromatography, the conjugate of HSA and Hb was obtained with the total yield about 50%. The P₅₀ and the Hill coefficient for the product were 16.1 mmHg and 1.82, respectively.     

The effect of solvated ions on the thermal denaturation of human serum albumin in water-dimethylsulfoxide solutions

The effects of solvated ions on the thermal denaturation of human serum albumin (HAS) in water-dimethylsulfoxide (DMSO) solutions were studied by the method of electron absorption spectroscopy. It was shown that depending on the DMSO concentration, electrolytes (LiCl, LiNO₃, LiClO₄, NaCl, and NaNO₃) contained in these solutions were characterized by different anion and cation solvation degrees: unlike cations, anions were only negligibly solvated, which affected HAS thermal denaturation. Electrostatic interactions between anions and positively charged amino acid residues supporting protein denaturation subsided in the line Cl⁻ > NO₃⁻ > ClO₄⁻.     

Transport of butyrate across the isolated bovine rumen epithelium--interaction with sodium, chloride and bicarbonate

The Ussing chamber technique was used for studying unidirectional fluxes of 14C-butyrate across the bovine rumen epithelium in vitro. Significant amounts of butyrate were absorbed across the bovine rumen epithelium in vitro, without any external driving force. The paracellular pathway was quantitatively insignificant. The transcellular pathway was predominately voltage-insensitive. The serosal to mucosal (SM) pathway was regulated by mass action, whereas the mucosal to serosal (MS) pathway further includes a saturable process, which accounted for 30 to 55% of the MS flux. The studied transport process for 14C-butyrate across the epithelium could include metabolic processes and transport of 14C-labelled butyrate metabolites. The transport of butyrate interacted with Na+, Cl- and HCO3-, and there was a linear relationship between butyrate and sodium net transport. Lowering the sodium concentration from 140 to 10 mmol l-1 decreased the butyrate MS flux significantly. Amiloride (1 mmol l-1) did, however, not reduce the butyrate flux significantly. Chloride concentration in itself did not seem to influence the transport of butyrate, but chloride-free conditions tended to increase the MS and SM flux of butyrate by a DIDS-sensitive pathway. DIDS (bilateral 0.5 mmol l-1) did further decrease the butyrate SM flux significantly at all chloride concentrations. Removing bicarbonate from the experimental solutions decreased the MS and increased the SM flux of butyrate significantly, and abolished net butyrate flux. There were no significant effects of the carbonic anhydrase inhibitor Acetazolamide (bilateral 1.0 mmol l-1). The results can be explained by a model where butyrate and butyrate metabolites are transported both by passive diffusion and by an electroneutral anion-exchange with bicarbonate. The model couples sodium and butyrate via CO2 from metabolism of butyrate, and intracellular pH.     

Intracellular activities of chloride, potassium and sodium ions in rabbit corneal epithelium

The mechanism of ion transport in the epithelium of rabbit cornea was studied by determining the intracellular ion activity of Cl-, Na+ and K+ under various conditions. Ionic activities were measured by means of microelectrodes containing liquid ion-exchangers selective for Cl-, Na+ or K+. The Cl- activity in basal cells of the epithelium in Na+ containing bathing solutions amounts to 28 +/- 2 mM (n = 11). This value is 1.9-times greater than expected on the basis of passive distribution across the tear side membrane. This finding suggests the existence of a Cl- accumulating process. Replacement of Na+ in the aqueous bathing solution by choline or tetraethylammonium results in a reversible decrease in Cl- activity to 22 +/- 1 mM (n = 11, P less than 0.025). The ratio of observed and predicted Cl- activity decreased significantly from 1.9 to 1.4 (P less than 0.05). The decrease in Cl- activity due to Na+ replacement was rather slow. In contrast, after readmittance of Na+ to the aqueous bathing solution, Cl- activity rose to a stable level within 30 min. These results indicate involvement of Na+ in Cl- accumulation into the basal cells of the epithelium. The K+ and Na+ activities of the basal cells of rabbit corneal epithelium in control bathing solutions were 75 +/- 4 mM (n = 13) and 24 +/- 3 mM (n = 12), respectively. The results can be summarized in the following model for Cl- transport across corneal epithelium. Cl- is accumulated in the basal cells across the aqueous side membrane, energized by a favourable Na+ gradient. Cl- will subsequently leak out across the tear side membranes. Na+ is extruded again across the aqueous side membrane of the epithelium by the (Na+ + K+)-ATPase.     

Effects of sodium, potassium and chloride ions on the membrane potential of Valonia aegagropila

Changes of the resting potential of Valonia cell in sea wateragainst a 10-fold increase of the external concentrations ofK^$, Na^$ and Cl^– were 1±1, 6.2±0.1 and 38.9±4mV, respectively. The potassium conductance was smaller than7 µ/cm², while the Na and Cl conductances were 45 and281 µ/cm², respectively, in normal sea water. The positivevacuolar potential could be explained by these ionic conductances.On the other hand, the membrane became more sensitive to K^$,if the cell was incubated for about 30 min in K-rich (100 mM)sea water. It is worth noting, however, that the membrane conductancewas lower in the K-rich sea water than in the normal sea water. (Received October 7, 1974; )     

The effects of sodium and potassium on ouabain binding by human erythrocytes

Ouabain binding by the human erythrocyte membrane is reversible, exhibits a high degree of chemical specificity, and can be detected at ouabain concentrations as low as 1 x 10^-10 M. The relation between ouabain binding and ouabain concentration can be described by a rectangular hyperbola permitting determination of the maximal binding (B _max) and the ouabain concentration at which ouabain binding is half-maximal (K_B). Reducing the external sodium concentration increased K_B, while reducing the external potassium concentration decreased K_B. Neither cation altered B _max The reciprocal of K_B was a linear function of the sodium concentration at sodium concentrations ranging from 0 to 150 mM. Conversely, the relation between the reciprocal of K_B and the external potassium concentration was nonlinear, and raising the potassium concentration above 4 mM produced no further increase in K_B. These results are compatible with a model which postulates that the erythrocyte membrane contains a finite number of receptors each composed of a glycoside-binding site and a cation-binding site. When sodium occupies the cation-binding site, the affinity of the glycoside site for ouabain is increased; when potassium occupies the cation-binding site the affinity of the glycoside site for ouabain is decreased.     

The subsequent effect of interaction between Co(2+) and human serum albumin or bovine serum albumin.

A notable hysteretic effect has been observed in the interaction of Co(II) with human serum albumin (HSA) or bovine serum albumin (BSA) using UV-Visible spectrometry at physiological pH (7.43), which shows that the binding between Co(II) and HSA or BSA may induce a slow transition of HSA or BSA from the conformation of weaker affinity for Co(II) to one of stronger affinity (A-B transition). The rate constants and activation parameters of this transition were measured and are discussed. It is inferred that such a conformation transition may occur due to the binding of the first Co(II) ion with the peptide segment of N-terminal residues 1-3, which results in a 'hinged movement' of the relatively hydrophobic 'valley' in the IA subdomain. This process leads to a slow conformational transition in the albumins, makes the other binding sites of Co(II) exposed, and shows a positive cooperativity effect. The LMCT (ligand-to-metal charge transition) bands of the Co(II)-HSA and Co(II)-BSA systems also show a kind of hypochromic effect featuring a dipole-dipole interaction mechanism. This phenomenon is rarely reported.     

Bovine serum albumin partitioning in an aqueous two-phase system: Effect of pH and sodium chloride concentration

The partitioning of bovine serum albumin (BSA) in a polyethylene glycol 3350 (8% w/w)–dextran 37 500 (6% w/w)–0.05 M phosphate aqueous two-phase was investigated at different pHs, at varying concentrations of sodium chloride at 20°C. The effect of NaCl concentration on the partition coefficient of BSA was studied for the PEG–dx systems with initial pH values of 4.2, 5.0, 7.0, 9.0, and 9.8. The NaCl concentrations in the phase systems with constant pH value were 0.06, 0.1, 0.2, 0.3, and 0.34 M. It was observed that the BSA partition coefficient decreased at concentrations smaller than 0.2 M NaCl and increased at concentrations greater than 0.2 M NaCl for all systems with initial pHs of 4.2, 5.0, 7.0, 9.0, and 9.8. It was also seen that the partition coefficient of BSA decreased as the pH of the aqueous two-phase systems increased at any NaCl salt concentration studied.     

Haemolysis of various mammalian erythrocytes in sodium chloride, glucose and phosphate-buffer solutions.

Mouse, rat, rabbit, hamster, cow, pig, sheep, guinea-pig, dog and human erythrocytes were studied. A 0.9% or stronger solution of sodium chloride completely prevented haemolysis; sheep and pig erythrocytes appeared the more fragile, while human and dog erythrocytes were not haemolized in concentrations of 0.4% or more. Haemolysis of human, rabbit, cow, hamster, guineapig, pig and sheep erythrocytes was not observed in solutions of 0.4% or more of glucose. Except for sheep, human and dog erythrocytes, haemolysis was depressed in rate but not completely prevented by phosphate-buffer solution of pH 7.0.     

The pH dependence of exchange transport of glucose in human erythrocytes

In glucose exchange transport into red blood cells the rate of glucose uptake showed two pH dependent maxima, with the larger at approximately pH 7.5 and the smaller one at pH 3. In the studied pH range the relation between the rate of glucose uptake and the substrate concentration followed Michaelis-Menten kinetics. While the maximal velocity (V) reflected the pH changes of the media, the Michaelis constant (K_m) remained constant. The dissociation constants of the groups of the free carrier and the carrier-glucose complex were the same. The pK of the acidic group was 5.2 and of the basic group 9.5. Glucose was not bound to groups of the carrier which dissociated protons in the pH range of three to nine. By rearranging the equation for the pH dependence of the relative influx a more definitive graphic determination of the pK values was produced.